Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo

View article at: http://www.nature.com/nm/journal/v7/n9/pdf/nm0901-1035.pdf
Download this as a file

The objective of this research is to create small diameter blood vesselswith endothelial progenitor cells (EPCs) that circulate in the blood. EPCs from peripheral blood are an endothelial cell source and typicallymigrate to regions with injured endothelia. What makes EPCs unique is itsability to produce nitric oxide, a vasodilator, and promote patency. Previously, in vitro culture of smooth muscles and endothelial cells onbiodegradable or biological scaffold material were used to produce avessel, but these grafts often times developed thrombosis, aggregation of blood factors that causes vascular obstruction, shortly after implantation.

EPCs were first isolated from blood samples from 1 to 2 week old lambs,cultured ex vivo, and then seeded onto decellularized vascular vesselsprepared from porcine iliac arteries 4 mm in diameter and 4-5 cm inlength. After seeding, grafts were preconditioned with variations ofshear stress in order to achieve maximal retention. The production ofnitric oxide was then assessed using an organ chamber. When exposed tocalcium inonphore A23187, grafts not seeded with EPCs remained contractedwhile grafts seeded with EPCs relaxed. This shows that EPC-seeded graftsproduced nitric oxide. EPC-seeded grafts were then implanted into sheepand data shows that all grafts seeded with EPCs were fully patent 130 daysafter implantation, whereas non-seeded EPC grafts occluded within 15 days. Due to the EPC-seeded grafts’ ability to contract and produce nitricoxide proves that EPCs are ideal in creating small-diameter vessels.

I chose this article because it is relevant and applicable to a large majority of the population since cardiovascular disease continues to be the leading cause of death in the US. It's also valuable to know that studies are being done to provide alternative, non-invasive treatments for cardiovascular disease.

Autologous Human Tissue-Engineered Heart Valves: Prospects for Systemic Application

To view the article, visit http://gateway.ut.ovid.com/gw1/ovidweb.cgi and type in the above title.

Past efforts on heart valve replacement have concentrated on the development of pulmonary (to the lungs) rather than systemic valves and the resultant valves have also proved unable to properly grow, adapt, and heal. In their July 2006 article, Anita et al. successfully incorporated several recently published techniques of cell seeding using fibrin gel (as a cell carrier), cell culture on a biodegradable scaffold, and mechanical cell conditioning to induce more anisotropy and valve-like behavior. Cells simply cultured statically were compared with cells subject mechanical conditioning in a bioreactor that simulates blood flow in a human heart, especially during diastole (when the heart dilates as it is filled with blood).

The original cells were harvested from the vena saphena magma of a 77 year old man and then isolated using an automated cell staining system and three primary antibodies. The trileaflet heart valves were grown into the correct shape using a mold, stents to hold up each leaflet, and scaffolds that had been sterilized with 70% ethanol. These cells were allowed to grow with media which among other components, contained 10% fetal bovine serum. Cells were than either a.)simply left to grow on culture flasks, b.) simply left to grow in the aforementioned bioreactor, and c.) grown in the bioreactor mimicking the heart’s diastolic phase with pressure differences of 5 to 30 mm Hg. The cells in group c.) were analyzed after 2, 4, and 6 weeks while the a.) and b.) cells were analyzed at the 4 week mark only.

While the conditioning proved to have little effect on cell growth or collagen content, it did succeed in generating tissue with more uniform mechanical properties than the statically grown controls. While the tissue engineered valves were able to generate similar stress strain curves in the radial direction as compared real heart valves, they performed less strongly in the circumferential direction. This most likely contributed to a larger problem - the valves opened correctly but failed to close completely, allowing blood to flow in the opposite direction. While further efforts must obviously be made in perfecting this technique, this new method of valve replacement culture shows great promise.

I first chose this article for its creative application of recent advances to tailor and design a new way to create a sorely needed tissue engineered device; reading widely and applying previously proven results to our project design is something we could all come into the habit of doing as engineers. The methods of tissue culture and testing were too numerous to mention completely, but this article is a great example of a large scale combination of various analytic tools we have learned about in class including histology, recognition using antibodies, mechanical stress/strain testing, cell counting by dyes to determine DNA amount (similar to our technique of nuclear staining with DAPI).

(Terry sez: a direct link to the paper can be found here - http://circ.ahajournals.org/cgi/content/full/114/1_suppl/I-152)

Tissue engineered cartilage: Utilization of autologous serum and serum-free media for chondrocyte culture

The main focus of this study is to examine the possible use of autologous serum and serum free media in chondrocyte growth for tissue engineered cartilage. They focus on the the different rates of proliferation of chondrocytes between the media with autologous serum, serum free media and the media with standard fetal bovine serum (FBS).

Chondrocytes were first isolated by taking auricular cartilage from pigs and fragmenting the sample. The sample was then washed, digested with 0.3% collagenase II, and filtered. The filtrate was taken and cell pelleted for use in the experiment. The chondrocytes were plated and the sample was placed into three different flasks with equal concentrations. The chondrocytes were subjected to three different media: Ham F12 culture media with 10% FBS, Ham F12 culture media with 10% autologous serum and Ham F12 culture media with growth factors. The culture was changed twice a week with morphology examined using light microscopy. The cells were trypsinized at days 3, 6, 9, and 12 with 0.25% Trysin/EDTA and counted with a hematocytometer.

The results showed that while the cells in the media with FBS grew faster initially, the cells in the serum-free media eventually caught up to the same cell count while the cells in the media with autologous serum grew at a slower rate. These findings prove that it is possible for chondrocyte expansion using media with autologous serum and serum-free media. The potential of these results is that the use of different media could reduce possible immunological reactions or vector infections from the FBS.

I chose this paper due to the similarities with our current labs involving Rex and 3T3 cells. In our lab we determined that the calf serum we were using was possibly influencing our results by containing some proteins that were analyzed in our quantification of protein. By using the serum-free media we could have nullified the serum's effects. Also, this directly applies to our goals of TE cartilage. Lastly, I feel that this paper illustrates various techniques we learned in class such as passaging and cell counting.

Tissue-Engineered Human Vascular Media Produced in Vitro by the Self-Assembly Approach Present Functional Properties Similar to Those of Their Native Blood Vessels

See the full text in http://www.liebertonline.com/doi/abs/10.1089/ten.2006.12.2275


The main object for their research is to develop vascular models which can be use for experimental studies in vitro. They are focusing on reconstruct the tissue-engineered vascular media, which is a very good in vitro model for the study of the functionality of human blood vessels.

The way to do this is first obtaining the endothelial cells from human umbilical vein. They test the responds of the human umbilical veins to endothelial cells(ET), and they figure out that by the activation of the ET receptors, the veins are able to responses to the ET. They then use the vascular smooth muscle cell to reconstruct the tissue-engineered vascular media. They first isolated the smooth muscle cell from the umbilical cord veins, and then culture the cell from human umbilical cord veins which has the expression on both the ET receptors. Then the smooth muscle cells are used to reconstruct the cultured media. By using the RT-PCR, they find out that there is mRNA of the both the ET receptors inside the Media they just reconstructed. By comparing the result with other publishes, the media shows the similar response to ET as the blood vessel from the smooth muscle cells.

Human blood vessels have very complex structures and they are not easy to take care. Therefore, doing experiments on human blood vessels to study the blood vessel functionality are like impossible work. For this reason, people want to use models rather than using the real blood vessels to the relative research. I like this article, because it gives idea on the development of blood vessels model. They can reconstruct the vascular media by using the tissue-engineered methods: isolates the vascular smooth muscle cells from human umbilical cord veins. They also test the response of the vascular media to blood vascular cells, and confirm that the media have similar kinds of response as the regular blood vessel.

http://www3.interscience.wiley.com/cgi-bin/fulltext/113391970/HTMLSTART

Growing tissue-like constructs with Hep3B/HepG2 liver cells on PHBV microspheres of different sizes

In this paper, the researchers were interested in the molecular, functional, and proliferative behaviors of liver cells, from the Hep3B and HepG2 hepatoma cell lines, seeded on 8% poly[3-hydroxybutyrate-co-3-hydroxyvalerate] (PHBV) microspheres as compared to the liver cells cultured 2-dimentionally with laminin and poly-L-lysine (positive control). This new cell seeding technique could become a possible cell growing methods in a tissue engineered liver/liver device. They choose PHBV because it is biocompatible, biodegradable, and non-cytotoxic in vivo. The microspheres were made by emulsifying and homogenizing 8% PHBV oil-in-water-in-oil solution, followed by evaporation of the solvent. The researchers considered microsphere size as a parameter and made 3 different sizes of microsphere by changing the homogenizer’s speed. After the microspheres were made, hepatoma cells were seeded onto them and cultured. As positive control, hepatoma cells are cultured two dimensionally with laminin and poly-L-lysine. Polyurethane film with zinc diethydithiocarbamate was used as negative control. At the appropriate time points, the cultured is analyzed. The analysis includes MTT assay for viability, bisbenzimide fluorescent assay for cell proliferation, ELISA assay to quantify albumin (since healthy liver cells make albumin), and EROD assay to test cytochrome P-405 activity (which measure liver cell’s detoxification activity).

The researchers were able to show that cells cultured on the microsphere spread over the microsphere and connect the adjacent microspheres together. There were multiple cells layer formed in the space between the microspheres. More interesting, it was shown that cells seeded on the microsphere had higher proliferation rate and viability, secreted more albumin, and had higher detoxification (cytochrome P-405) activity than the positive control. This means that the cells are able to live longer, divide better, and maintain liver specific activities/functions better. These behaviors of cells cultured on microsphere made this new culturing method a good choice to use in a tissue-engineered liver or liver device.

The reasons I chose the paper are as followed. (1) Liver is a very important organ and many people are waiting for the limited liver transplantation. This paper is attempting to solve this problem by researching tissue engineered liver. (2) Many of the finding in this paper will be useful for a tissue engineered (TE) liver. For example, liver cells grow on microsphere proliferate better and have higher viability. This is extremely important since primary cells used in TE liver are limited, usually not very proliferative, and their viability decrease with time. This new technique could improve, if not solve, these problems. Livers cells seeded on the microsphere secreted higher albumin and have higher P-405 activity, suggesting that they maintain their function and not likely to de-differentiate. This aspect is especially useful in TE liver since we want the cells to maintain their function instead of ceasing to act like liver cells. (3) I think it’s really nice that this paper use many of the methods we learned in class such as trypan blue plus other live/dead assay, and ELISA. I picked this paper because the materials are relevant to the class and tissue engineering.

Autologous full-thickness skin substitute for healing chronic wounds

The paper’s objective was to develop tissue engineered skin with human origin in order to use to heal wounds. The paper focused on the development of skin in order to heal ulcers. To develop the tissue engineered skin, the investigators harvested a 3-mm biopsy from the patient’s leg. From this sample of human skin, they prepared an acellular human allodermis. Dermal fibroblasts were cultured along with epidermal sheets. The next step was placing epidermal sheets stratum corneum side up onto the allodermis and then culturned in keratinocyte medium for approximately 7 days. The allodermis with the epidermal sheets were then placed on the fibroblasts to allow for migration. With further culturing and addition of growth factors, antibiotics, and growth medium, the tissue engineered skin was ready in three weeks. Within the three week period the epidermis expanded to cover to allordermis, with a surface area of 1.5 cm2 from the original 3-mm biopsy. Like human skin, the engineered skin was comprised of a compact basal layer, spinous layer, granular layer, and statum corneum.

The engineered skin was applied to the skin using Lomateull® H and providone-iodine ointment. The engineered skin was used to treat nineteen ulcers in fourteen patients. The use of engineered skin resulted in pain reduction from the ulcers. Within one week of application, the engineered skin expanded to the surrounding skin of the ulcer. In twelve of the nineteen ulcers, the engineered skin was incorporated into the wound. The median time for healing was 6 weeks. However, in seven of the nineteen ulcers, the skin was not incorporated and could be removed from the wound.

I choose this paper because it demonstrates the effectiveness of using tissue engineered skin in order to treat wounds. This paper shows that a small sample of human skin can be used to create an effective treatment. Tissue engineered skin is an important area of research and development to help the myriad of skin conditions and also treating severe damage, like burns. Although three weeks is a long time to develop the skin, it is a great alternative to skin grafts where a large area of healthy skin is needed.

Gibbs, S., van den Hoogenband, H.M., Kirtschig, G., Richters, C.D., Spiekstra, S.W., Breetveld, M., Scheper, R.J., and de Boer, E.M. “Autologous full-thickness skin substitute for healing chronic wounds.” British Journal of Dermatology 155 (2006): 267-274.

PubMed Link:
Click Here


Full Text Link (UC-elinks):
http://www.blackwell-synergy.com/doi/abs/10.1111/j.1365-2133.2006.07266.x